Gene therapy broadly involves the use of genetic material to treat disease. It includes the supplementation in cells with defective genes (e.g. those harbouring mutations) with functional copies of those genes, the inactivation of improperly functioning genes and the introduction of new therapeutic genes.
Therapeutic genetic material may be incorporated into the target cells of a host using vectors to enable the transfer of nucleic acids. Such vectors can be generally divided into viral and non-viral categories.
Viruses naturally introduce their genetic material into target cells of a host as part of their replication cycle. Engineered viral vectors harness this ability to enable the delivery of a nucleotide of interest (NOI) to a target cell. To date, a number of viruses have been engineered as vectors for gene therapy. These include retroviruses, adenoviruses (AdV), adeno-associated viruses (AAV), herpes simplex viruses (HSV) and vaccinia viruses.
In addition to modification to carry the nucleotide of interest, viral vectors are typically further engineered to be replication defective. As such, the recombinant vectors can directly infect a target cell, but are incapable of producing further generations of infective virions. Other types of viral vectors may be conditionally replication competent within cancer cells only, and may additionally encode a toxic transgene or pro-enzyme.
Retroviral vectors have been developed as therapies for various genetic disorders and are now showing increasing promise in clinical trials (e.g. Galy, A. and A. J. Thrasher (2010) Curr Opin Allergy Clin Immunol 11(6): 545-550; Porter, D. L., B. L. Levine, M. Kalos, A. Bagg and C. H. June (2011) N Engl J Med 365(8): 725-733; Campochiaro, P. A. (2012) Gene Ther 19(2): 121-126; Cartier, N., S. Hacein-Bey-Abina, C. C. Bartholomae, P. Bougneres, M. Schmidt, C. V. Kalle, A. Fischer, M. Cavazzana-Calvo and P. Aubourg (2012) Methods Enzymol 507: 187-198; Sadelain, M., I. Riviere, X. Wang, F. Boulad, S. Prockop, P. Giardina, A. Maggio, R. Galanello, F. Locatelli and E. Yannaki (2010) Ann N Y Acad Sci 1202: 52-58; DiGiusto, D. L., A. Krishnan, L. Li, H. Li, S. Li, A. Rao, S. Mi, P. Yam, S. Stinson, M. Kalos, J. Alvarnas, S. F. Lacey, J. K. Yee, M. Li, L. Couture, D. Hsu, S. J. Forman, J. J. Rossi and J. A. Zaia (2010) Sci Transl Med 2(36): 36ra43 and Segura M M, M. M., Gaillet B, Gamier A. (2013) Expert opinion in biological therapy).
Important examples of such vectors include the gamma-retrovirus vector system (based on MMLV), the primate lentivirus vector system (based on HIV-1) and the non-primate lentivirus vector system (based on EIAV).
Reverse genetics has allowed these virus-based vectors to be heavily engineered such that vectors encoding large heterologous sequences (circa 10 kb) can be produced by transfection of mammalian cells with appropriate DNA sequences (reviewed in Bannert, K. (2010) Caister Academic Press: 347-370).
Engineering and use of retroviral vectors at the research stage typically involves the production of reporter-gene vectors encoding, for example, GFP or lacZ. The titres of these clinically irrelevant vectors are usually in the region of 1×106 to 1×107 transducing units per mL (TU/mL) of crude harvest material. Further concentration and purification of this material can achieve working stocks in excess of 1×1010 TU/mL. However, the production of vectors encoding therapeutically relevant NOIs often results in substantially reduced titres compared to these reporter vectors.
There are several factors that are potentially responsible for this effect:                1. The size of the therapeutic genome. Very large genomes can be packaged by retroviruses, but it is thought that reverse transcription and/or integration steps become less efficient as size increases.        2. The stability of vector genome RNA. This may be reduced by the presence of unpredicted instability elements in the NOI.        3. Suboptimal nucleotide usage within the vector genome RNA. Wild-type virus genomes often have a certain nucleotide bias (e.g. HIV-1 is AT rich). Vector genomes tend to be less AT rich, which may affect packaging and/or post-maturation steps.        4. Expression of the NOI in viral vector production cells. (Over-)expressed protein may have an indirect or direct effect on vector virion assembly and/or infectivity.        
We have empirically shown that expression of the protein encoded by the NOI within viral vector production cells can adversely affect therapeutic vector titres (see FIGS. 3i and 3ii).
Incorporation of a protein encoded by the NOI (the protein of interest, POI) into vector virions may also impact downstream processing of vector particles; for example, an NOI encoding a transmembrane POI may lead to high surface expression of the transmembrane protein in the viral vector virion, potentially altering the physical properties of the virions. Furthermore, this incorporation may present the POI to the patient's immune system at the site of delivery, which may negatively impact transduction and/or the long-term expression of the therapeutic gene in vivo. The NOI could also induce the production of undesirable secondary proteins or metabolites which could impact production, purification, recovery and immunogenicity and it is therefore desirable to minimise this.
There is a clear need for the capability to repress the expression of a NOI in viral vector production cells while maintaining effective expression of the NOI in target cells. Whatever mechanism is employed, the ‘natural’ pathway of assembly and resulting functionality of the viral vector particles must not be impeded. This is not straightforward because the viral vector genome molecule that will be packaged into virions must necessarily encode the NOI expression cassette. In other words, because the vector genome molecule and NOI expression cassettes are operably linked, modification of the NOI expression cassette may have adverse consequences on the ability to produce the vector genome molecule in the cell. For example, if a physical transcription block (e.g. TetR repressor system) is used to repress the NOI expression cassette it is likely that production of the vector genome molecule would also be inhibited through steric hindrance. In addition, control mechanism modifications must also not adversely affect the functionality of the vector genome molecule after virion maturation and release (i.e. with regards to directing transduction of the target cell). For example, a retroviral vector genome RNA molecule must be capable of the processes of reverse transcription and integration—any modification to the NOI expression cassette must not impede these steps in the transduction process.
Repression of NOI expression within viral vector production cells may present further advantages. If NOI expression leads to a reduction in the viability of vector production cells, its repression may benefit manufacturing at large scale which requires large cell numbers. The reduction in cell debris due to cell death would also reduce impurities within the crude vector harvest material. Processing, purification and concentration of the vector platform (i.e. different therapeutic genes encoded within the same vector system) could be standardised; if the only heterologous genes expressed within viral vector production cells are those required for vector production, downstream processing could be more easily optimised for an entire platform of therapeutic vectors, resulting in very similar physical specifications of vector preparations. Variability of immune response to, and toxicity of, resulting vectors in vivo may be minimised, which may lead to more persistent therapeutic NOI expression in the target cells.
Tissue-specific promoters which limit expression of the NOI in production cells are a possible solution to this problem, although leakiness of these promoters might lead to adverse levels of transgene protein. However, greater and more robust expression of the NOI in target cells can be achieved using constitutive promoters. Indeed, such robust expression may be required for efficacy in vivo. In addition, tissue-specific promoters may be less predictable when following a therapeutic vector product through animal models and into humans during pre-clinical and clinical development.